Plasmon-Enhanced Imaging of Single Membrane Proteins

单膜蛋白的等离激元增强成像

• 批准号: 0930074
• 负责人: Lukas Novotny
• 金额: $ 39.97万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0930074&HistoricalAwards=false
• 关键词: Plasmon; Enhanced; Imaging; Single; Membrane

0930074NovotnyThis proposal seeks to fundamentally improve the resolution of optical imaging by exploiting the properties of optical antennas, a new concept in the field of optical physics. The objective is to develop an instrument for the imaging, identification and characterization of single proteins in native cell membranes. An instrument with such capability will have a high impact on various fields of biological research because membrane proteins undergo a multitude of specific tasks ranging from recognition and signaling to molecular transport across the cell membrane. Although current methods, such as X-ray crystallography or NMR, provide high-resolution structural details, they are limited by their static observation and ensemble averaging. As a consequence, the spatial distribution and co-localization of membrane proteins as well as the correlations with membrane topology are largely unknown. The instrument to be developed in this project uses an optical antenna to localize and enhance incident laser radiation thereby creating a nanoscale light source. This light source will be rasterscanned over the surface of a biological membrane with fluorescently labeled proteins. Each point on the sample surface will be characterized by its unique fingerprint in form of a fluorescence spectrum allowing us to localize, identify and study single proteins. Preliminary results support the feasibility of this technique but various issues need yet to be addressed. We will use fused gold nanoparticles and gold nanorods as optical half-wave antennas and attach them to pointed dielectric capillaries. Different approaches for suppression of background fluorescence originating from the cell interior will be investigated. The instrument will be used to study distributions of adhesion proteins in endothelial cell membranes which play a key role in leukocyte-endothelial cell interactions and thus in the inflammatory response. Single layers of endothelial cells will be grown directly on functionalized glass substrates and P-selectin proteins will labeled with fluorescent antibodies. The goal is to spatially resolve individual proteins under physiological conditions and to study the correlation with membrane topology.

0930074 Novotny该提案旨在通过利用光学天线的特性从根本上提高光学成像的分辨率，这是光学物理学领域的一个新概念。我们的目标是开发一种仪器，用于成像，识别和表征天然细胞膜中的单个蛋白质。具有这种能力的仪器将对生物学研究的各个领域产生重大影响，因为膜蛋白经历了从识别和信号传导到分子跨细胞膜运输的多种特定任务。虽然目前的方法，如X射线晶体学或NMR，提供了高分辨率的结构细节，它们受到静态观察和系综平均的限制。因此，膜蛋白的空间分布和共定位以及与膜拓扑结构的相关性在很大程度上是未知的。在这个项目中开发的仪器使用光学天线来定位和增强入射激光辐射，从而创建一个纳米级光源。该光源将在具有荧光标记的蛋白质的生物膜的表面上进行光栅扫描。样品表面上的每个点都将以荧光光谱的形式通过其独特的指纹进行表征，从而使我们能够定位，识别和研究单个蛋白质。初步结果支持这种技术的可行性，但各种问题还有待解决。我们将使用熔融的金纳米颗粒和金纳米棒作为光学半波天线，并将它们连接到尖介电毛细管上。将研究用于抑制源自细胞内部的背景荧光的不同方法。该仪器将用于研究粘附蛋白在内皮细胞膜中的分布，其在白细胞-内皮细胞相互作用中起关键作用，从而在炎症反应中起关键作用。单层内皮细胞将直接生长在功能化的玻璃基底上，P-选择素蛋白将用荧光抗体标记。目标是在生理条件下空间分辨单个蛋白质，并研究与膜拓扑结构的相关性。